Abstract - A large body of evidence exists which implicates enterotoxin-producing Escherichia coli as the cause of high percentage of acute diarrhea in humans. Studies by veterinarian microbiologists have provided firm evidence that certain plasmid-mediated surface antigens allow toxigenic E. coli to colonize the small bowel mucosa and cause diarrheal diseases in young animals. The objective of this research is to define the genetic and physiologic control of adherence antigens type 1,2,3. Restriction-ligation techniques will be employed to clone plasmid DNA regions mediating production of our three immunologically distinct adherence antigens. Once clone, these DNA sequences can be compred by heterodupic analysis and/or Southern blots for homology. Plasmid-mediated synthesis of the antigenic surface adherence antigens will be documented in E. coli minicells. An alternative genetic approach to "label" cryptic plasmids controlling pili production will involve infection with a coliphae carrying the transposable element Tn5. Insertion of the Kanamycin resistance gene will greatly facilitate genetic studies of plasmids in ETEC strains. Novel adherence antigens on ETEC strains pathogenic for humans will undergo serologic, electron microscopic studies, and molecular weight determinations by SDS-PAGE. ETEC strains will be compared for their abilities to adhere to human buccal cells and human ileal cells. Should the latter assay prove to be a sensitive model for detection of ETEC pathogenic for man, further studies will include binding by labeled isolated pili, scanning electron microscopy and determination of the eukaryotic receptors for ETEC binding. Genetically-derived adherence antigen+, toxin-negative strains and ETEC strains with novel adherence antigens will be fed to human volunteers in order to assess the colonization ability of these two groups of strains with a long range aim toward development of vaccine strain(s). The studies proposed in this grant are designed to advance the level of knowledge concerning the complex interactions that take place in order for bacteria to stick to eukaryotic cell mucosal surfaces.